Human papillomavirus L2 facilitates viral escape from late endosomes via sorting nexin 17

Abstract

The human papillomavirus (HPV) L2 capsid protein plays an essential role during the early stages of viral infection, but the molecular mechanisms underlying its mode of action remain obscure. Using a proteomic approach, we have identified the adaptor protein, sorting nexin 17 (SNX17) as a strong interacting partner of HPV L2. This interaction occurs through a highly conserved SNX17 consensus binding motif, which is present in the majority of HPV L2 proteins analysed. Using mutants of L2 defective for SNX17 interaction, or siRNA ablation of SNX17 expression, we demonstrate that the interaction between L2 and SNX17 is essential for viral infection. Furthermore, loss of the L2-SNX17 interaction results in enhanced turnover of the L2 protein and decreased stability of the viral capsids, and concomitantly, there is a dramatic decrease in the efficiency with which viral genomes transit to the nucleus. Indeed, using a range of endosomal and lysosomal markers, we show that capsids defective in their capacity to bind SNX17 transit much more rapidly to the lysosomal compartment. These results demonstrate that the L2-SNX17 interaction is essential for viral infection and facilitates the escape of the L2-DNA complex from the late endosomal/lysosomal compartments.

Figure 1. HPV-16 L2 protein interacts with SNX17 in vitro and in vivo

(A) HEK293 cells were transfected with a plasmid expressing FLAG-HA-tagged HPV-16 L2. After 24 hr, extracts were immunoprecipitated using anti-HA antibody-conjugated agarose beads and the total immunoprecipitates were subjected to mass spectrometric analysis. The table shows a selection of the prominent HPV-16 L2-specific hits, together with their E-value (the base-10 log of the expectation that the assignment is stochastic), the number of unique peptide sequences (#) and total number of tandem mass spectra that can be assigned to each protein. (B) The list of unique SNX17 peptides found in the mass spectra. (C) Ectopically expressed wild type FLAG-HA-tagged HPV-16 L2 was extracted from HEK293 cells and subjected to pull-down assays with GST-SNX17 fusion protein. Bound proteins and cell extracts were analyzed by immunoblotting using anti-HA (HPV-16 L2) antibodies. (D) Endogenous SNX17 was extracted from HEK293 cells and subjected to pull-down assays with GST-HPV-16 L2 and GST-HPV-16 L1 fusion proteins. Bound proteins and cell extracts were analyzed by immunoblotting using anti-SNX17 antibodies. Arrows on the Ponceau-stained gel indicate the bands corresponding to the purified GST-fusion proteins used in the pull down-assay. (E) Endogenous SNX17 was extracted from HEK293 cells and subjected to pull-down assays with GST-HPV-16 L2. GST immunoprecipitates were then subjected to extensive washing with 0.5%, 1% or 2% Triton X-100 in PBS. Bound proteins and cell extracts were analysed by immunoblotting using an anti-SNX17 antibody. In all the GST binding experiments, non-fused GST proteins were used as a negative control. Inputs represent 10% of the extracts used for the pull-down assays. Asterisks (*) denote a non-specific band recognised by the anti-HA antibody. Numbers at the gel borders are molecular sizes in kilodaltons.

Figure 2. Mapping of the HPV-16 L2 – SNX17 interacting domains

(A) Wild type HPV-16 L2 and two L2 mutants with mutations in the proposed SNX17 binding site (N254A and 254NPAY257AAAA) were transiently expressed in HEK293 cells. After 24 hr, proteins were extracted and subjected to a pull-down assay with GST-SNX17 fusion protein. Bound proteins and cell extracts were analyzed by immunoblotting using an anti-HA antibody. (B) Wild-type SNX17 and SNX17 truncated variants were in vitro translated and incubated with GST-HPV-16 L2 protein. Bound proteins and inputs were assessed by autoradiography and the input GST fusion proteins were visualised with Coomassie staining. In both sets of assays, non-fused GST proteins were used as a negative control and inputs corresponds to 10% of the protein that was used in the pull-down assays. Asterisks (*) denote a non-specific band recognised by the anti-HA antibody. Numbers at the gel borders are molecular sizes in kilodaltons.

Figure 3. Loss of SNX17 abolishes HPV-16 infection

(A) HaCaT cells were transfected with siSNX17, siCTRL, different amounts of SNX17 expression plasmid or mock transfected. After 48 hr, cells were exposed to HPV-16 PsVs carrying a luciferase reporter plasmid. (B) Wild-type HaCaT and stable SNX17 knock-down HaCaT (shSNX17 HaCaT) cells were exposed to HPV-16 PsVs carrying a luciferase reporter plasmid. (C) HaCaT cells were exposed either to wild-type HPV-16 PsVs or HPV-16 PsVs with the mutated SNX17 binding site in L2 (L2 N254A mutant), both carrying a luciferase expression plasmid. In all experiments, cells were lysed 48 hr post-infection and the level of luciferase activity was evaluated in triplicate by luminometry. Obtained values were corrected for background luminescence and normalised to mock transfected HaCaT cells (A), wild type HaCaT cells (B) or HaCaT cells infected with wild type HPV-16 PsVs (C). Immunoblots show the level of SNX17 expression (AB) and input of PsVs used for the infection (C, lower panel). Alpha actinin was used as a loading control. The agarose gel in C (upper panel) shows the level of pGL3 plasmid carrying the luc+ gene extracted from PsVs used in the infectivity assay. Neutralisation assay using anti-capsid H16.V5 antibodies was performed in order to assess the level of luciferase activity due to the non-encapsidated luciferase plasmid DNA (A). Results are expressed as the means ± SD of at least three independent experiments, and the corresponding P values are: * P<0.05, **P<0.01, ***P<0.001

Figure 4. SNX17 does not affect HPV-16 trafficking to early endosomes

Wild-type HaCaT and stable SNX17 knock-down HaCaT (shSNX17 HaCaT) cells were exposed to AF488-labeled HPV-16 PsVs (in green) for 1 hr at 4°C. Cells were then washed and incubated at 37°C for 30 min (A) and 2.5 hr (B). After the incubation, cells were fixed and stained for endogenous EEA-1 (in red). The micrographs are representative and show the mid-cell body/nucleus focal planes. Scale bar = 10 μm.

Figure 5. SNX17 affects late phases of the HPV-16 trafficking

Wild-type HaCaT and stable SNX17 knock-down HaCaT (shSNX17 HaCaT) cells were exposed to AF488-labeled HPV-16 PsVs (in green) for 1 hr at 4°C. Cells were then washed and incubated at 37°C for 30 min (A), 3 h (B), 8 h (C) and 16 h (D). (E) HaCaT cells were exposed to AF488-labeled HPV-16 PsVs wild type or HPV-16 L2-N254A (in green) and incubated at 37°C for 8 hr. After the incubation, cells were fixed and stained for endogenous LAMP-2 (in red). The micrographs are representative and show the mid-cell body/nucleus focal planes. Scale bar = 10 μm.

Figure 6. SNX17 is involved in transfer of viral DNA into the nucleus

Wild type HPV-16 PsVs and HPV-16 PsVs with the mutation in the proposed SNX17 binding site (L2 N254A mutant) were generated in the presence of 5-ethynyl-2′-deoxyuridine (EdU). HaCaT cells were exposed to wild type or mutant PsVs for 1 hr at 4°C. Cells were then washed and incubated at 37°C for 16 hr (A) or 22 hr (B). After the incubation, cells were fixed and processed first for the detection of EdU-labeled DNA (in red) and then stained with anti-PML antibodies (in green). The micrographs are representative and show the mid-cell body/nucleus focal planes. Nuclei are illustrated by dotted lines. Arrows show colocalisation of EdU-labeled DNA and PML staining. Scale bar = 10 μm.

Figure 7. SNX17 increases stability of HPV-16 L2 protein

(A) Wild-type HaCaT and stable SNX17 knock-down HaCaT (shSNX17 HaCaT) cells were exposed to HPV-16 PsVs for 1 hr at 4°C. Cells were then washed and incubated at 37°C for the indicated periods of time. Cell lysates were subjected to immunoblotting using anti-L1 and anti-L2 antibodies. Zero time points represent 30% of the total sample. Alpha actinin was used as a loading control. (B) HEK293 cells were transfected with siSNX17, siCTRL or mock transfected. After 48 hr, cells were additionally transfected with HPV-16 L2 protein alone or in combination with SNX17 (+SNX17) and were grown for another 24 hr before harvesting. Cell lysates were assessed using anti-HA (HPV-16 L2) and anti-SNX17 antibodies. Beta-galactosidase was used as an internal control for monitoring the transfection efficiency. Asterisks (*) denote a non-specific band recognised by anti-HA antibody. (C) HEK293 cells were transiently transfected either with wild-type HPV-16 L2 or with HPV-16 L2 constructs carrying mutations in the proposed SNX17 binding site (L2 N254A and L2 254NPAY257AAAA). After 24 hr, cells were washed and treated with cycloheximide (CHX) for 3 hr, 6 hr, or 9 hr. Cell lysates were subjected to immunoblotting using an anti-HA antibody. The level of L2 proteins was quantitated by densitometry, corrected for differences in transfection efficiency (expression of β-galactosidase) and normalised to α-globulin as an internal loading control. Results are expressed as the means ± SD of four independent experiments. Protein half lives represent values determined from the linear regression curves.